A critical gap in our knowledge of HIV remains the molecular role of the envelope transmembrane subunit, gp41, in HIV viral entry. Our long-term goal is to advance the structural studies of large membrane protein complexes and the concerted conformational changes through which they function. The objective of this proposal is to better define the mechanism that leads to productive viral membrane fusion mediated by HIV gp41. The rationale that underlies the research proposed herein is that the molecular role of gp41 in membrane fusion can be elucidated using the latest advances in labeling and analysis. Novel reagents have been developed that facilitate labeling of envelope and will allow the field to move forward. In order to test these new reagents in proof of concept situations we propose the following specific aims: Specific Aim 1. Determine the dynamics of the macromolecular complexes involved in HIV entry in live cells using a single particle fusion assay. As fusion occurs and post-fusion events proceed, we will measure the dynamics of fusion pore formation by monitoring envelope subunit separation and separation from the capsid core. Specific Aim 2. Quantify the trapped intermediate of HIV gp41 in the virus/cell context. We will use covalently reactive peptides that bind to the gp41 intermediate and abrogate function. We will quantify the amount of trapped intermediate as compared to total protein. The proposed research is significant because it will dramatically increase the structural knowledge of the role of envelope in the replication cycle in the cellular context. Advancements in labeling will produce information with the potential to reveal novel drug targets within envelope.